1. Field of the Invention
The present invention relates to a non-reducing saccharide-forming enzyme, a trehalose-releasing enzyme, and a process for producing saccharides using the enzymes.
2. Description of the Prior Art
Trehalose is a disaccharide consisting of two moles of glucose bound at their reducing residues, and is widely found in nature, for example, in microorganisms, fungi, algae, insects, Crustacea, etc. Since the saccharide has long been known as a useful saccharide substantially free of reducibility and having a satisfactory moisture-retaining action, it has been expected to use in extensive fields including foods, cosmetics, and pharmaceuticals. However, no efficient production of the saccharide was established, and this narrows the use of trehalose in spite of its outstanding expectation. Thus supply of trehalose in a lower cost is greatly expected.
As a proposal for such an expectation, the present inventors had already established a process for enzymatically producing trehalose from material starches through their energetic studies. The process is characterized by a step of subjecting reducing partial starch hydrolysates to the action of a non-reducing saccharide-forming enzyme, which forms a non-reducing saccharide having a trehalose structure as an end unit from reducing partial starch hydrolysates, and to the action of a trehalose-releasing enzyme which acts on a non-reducing saccharide having a trehalose structure as an end unit in order to hydrolyze and release trehalose from the rest of the non-reducing saccharide. These enzymes and processes thereof are disclosed in Japanese Patent Kokai Nos. 143,876/95, 213,283/95, 322,883/95, 298,880/95, 66,187/96, 66,188/96, 73,504/96, 84,586/96, and 336,388/96, applied for by the same applicant as the present invention. Thus, a low-cost production of trehalose was attained.
During the studies, they found an original finding that the non-reducing saccharide-forming enzyme can be applied for a novel production of non-reducing saccharides that can overcome conventional drawback residing in reducing partial starch hydrolysates. As a problem, reducing partial starch hydrolysates such as dextrins and maltooligosaccharides have advantageous features that they can be used as sweeteners and energy-supplementing saccharide sources, but as a demerit they are highly reactive with substances because of their reducibility and are susceptible to browning reaction when coexisted with amino acids and/or proteins and to readily deteriorate their quality. To overcome such a problem, it is only known a method to convert reducing partial starch hydrolysates into sugar alcohols using a high-pressure hydrogenation method, etc. In actual use, the method, however, needs much heats and instruments constructed under consideration of safety in view of the use of hydrogen, resulting in a higher cost and much labor cost. On the contrary, the aforesaid non-reducing saccharide-forming enzyme as mentioned previously acts on reducing partial starch hydrolysates and forms non-reducing saccharide having a trehalose structure as an end unit, and the reaction proceeds under a relatively-mild condition due to its enzymatic reaction. Using the action of the enzyme, the present inventors established a novel efficient process for non-reducing saccharides using the enzyme, that can overcome conventional drawback residing in reducing partial starch hydrolysates. Because of these findings, the development of applicable uses for trehalose and non-reducing saccharides have become to be flourished in various fields, and this diversifies the uses of these saccharides and now remarkably increases the demands of the saccharides in a wide variety of fields.
Under these circumstances, a more efficient process for producing trehalose and non-reducing saccharides having a trehalose structure has been more expected in this art. A key to such an expectation is to establish a non-reducing saccharide-forming enzyme and a trehalose-releasing enzyme with various optimum conditions, and to provide a wide variety of sources for such enzymes usable in the production of the saccharides. Thus, an optimum enzyme can be chosen from various types of enzymes depending on the optimum conditions of another enzymes usable in combination with the above enzymes to produce the desired saccharides, as well as on installations and final uses of the saccharides produced, resulting in an efficient production of the saccharides. Conventionally known non-reducing saccharide-forming enzymes can be grouped into those having optimum temperatures of relatively-lower temperatures of about 40° C. or lower, and those having optimum temperatures of relatively-higher temperatures of about 60° C. or higher. While conventionally known trehalose-releasing enzymes can be grouped into those having optimum temperatures in a relatively-lower temperature range, about 45° C. or lower, and those having optimum temperatures in a relatively-higher temperature range, about 60° C. or higher. However, any non-reducing saccharide-forming enzyme and a trehalose-releasing enzyme having an optimum temperature in a medium temperature range, about 50° C., have never yet been opened.
Among saccharide-related enzymes used in the production of saccharides from starch materials, enzymes as a major group have an optimum temperature in a medium temperature range. Such enzymes may be required in the process for producing the aforesaid trehalose and non-reducing saccharides; No non-reducing saccharide-forming enzyme and no trehalose-releasing enzyme, which have an optimum temperature in a medium temperature range, have not yet been established so that there has not yet been realized a process for producing saccharides in a sufficient yield using either or both of these enzymes together with the above saccharide-related enzymes. Depending on installations for producing saccharides and final uses of them, there have been required enzymes having an optimum temperature in a medium temperature range in their enzymatic reactions. It is far from saying that it has established a process for producing saccharides in a satisfactorily-high yield using a non-reducing saccharide-forming enzyme and a trehalose-releasing enzyme. As described above the establishment of a non-reducing saccharide-forming enzyme and a trehalose-releasing enzyme having an optimum temperature in a medium temperature range, and a process for producing saccharides comprising non-reducing saccharides are in great demand.